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ì Chlorophyll Analysis & Extrac/on
Chlorophyll a
Mg2+
Week 2 Experiment Summary
ì Spinach Extrac/on ì Grind w/ acetone, add hexane, then water. Separate layers &
add MgSO4 to hexane layer
ì TLC of Extract ì Developing Chamber ì Rf Calcula/on
ì Absorbance & Fluorescence Measurement of Extract ì Determina/on of [Chlorophyll] from A & F data
Which of the following is false?
A. Electronega/vity is an atom’s ability to aMract shared electrons to itself.
B. A bond dipole arises when a electronega/vity difference exists between two atoms.
C. A significant bond dipole exists between C-‐H in heptane.
D. CCl4 has a molecular dipole.
Polarity Continuum
H-‐H < H-‐S < H-‐Cl < H-‐O < H-‐F < Na -‐ F (2.1)(2.1) (2.1)(2.5) (2.1)(3.0) (2.1)(3.5) (2.1)(4.0) (0.9)(4.0)
δ+ δ-‐ δ+ δ-‐ δ-‐ δ-‐ δ+ δ+ + -
ΔEN
Dipole-‐Dipole Forces
ì Defini/on:
ì Energy compared to a covalent bond?
ì Sketch the dipole-‐dipole force that exists between two molecules of acetone.
electrosta/c aMrac/on between the par/ally posi/ve & nega/ve ends of polar molecules
Edip-‐dip = 1 kJ/mol (~1% covalent bond energy)
Hydrogen Bonding
ì Defini/on:
ì Energy compared to a covalent bond?
ì Sketch the hydrogen bonding force that exists between two molecules of water.
dipole-‐dipole forces for polar molecules with hydrogen atoms bound to oxygen, nitrogen, or fluorine atoms.
EHbond = 10 kJ/mol (~5% covalent bond energy)
London Dispersion Forces
Also known as (aka):
Induced Dipole Forces, van der Waals Forces
ì Defini/on (What happens?): ì Transitory dipole forma/on: random e-‐ shih results in δ+ &
δ-‐ ends of a molecule/atom ì Disturbing other molecules/ atoms to form more
transitory dipoles & weak electrosta/c aMrac/on
ì Sketch the Dispersion force that exists between four molecules of propane. (You’ll need 2 steps.)
Which of the following is false?
A. Hydrogen bonding occurs between molecules containing C-‐H bonds.
B. Dipole Dipole forces exist between water molecules.
C. Dispersion Forces exist between acetone molecules.
D. Intermolecular forces between nonpolar molecules decrease with molecular weight.
Why is Chlorophyll’s structure special?
Tail
ì nonpolar
ì saturated
Head
ì unsaturated & conjugated
ì planar
ì intermediate polarity
What chemicals are in ?
ì Chlorophyll a & b
ì Pheophy/n a & b
ì Xanthophylls
Extraction / Separatory Funnels
ì How do you use a separatory (sep) funnel?
ì What is the iden/ty of the layers?
ì Which layer contains “ ”?
ì What is MgSO4 used for?
Thin Layer Chromatography (TLC)
ì What is the TLC process?
ì Developing Chamber
ì Prep & Spot Plate
ì Develop Plate
ì Measure Rf = xorigintocpd xorigintofront
What are the Rfs for A, B, & C? Does the size of plate ma2er?
3 Classes of “Chemicals” Silica SiO2
ì Phase? Sta0onary
ì Polarity?
POLAR Si-‐O-‐H (silanol) groups on outside of silica par/cles
Eluent
ì Phase? Mobile ì Polarity? NONPOLAR to intermed. polar Eluent use wrt polarity? in order of ñpolarity
Use order of these eluents?acetone, CH3(CH2)4CH3, CH2Cl2, CH3CH2OCH2CH3
Analyte
ì Defini/on? Mixture of 2 or more chemicals to be separated
ì Polarity?
NONPOLAR to intermediate polar
Explain your answer.
How does TLC separate these chemicals?
By Polarity…
ì Is A polar, intermediate polar, or nonpolar?
ì Which chemical is the most strongly adsorbed to the silica plate? What can be said about its polarity?
ì Which eluent is most likely responsible for the separa/on seen to the right: pure acetone, 1:1 mix of acetone & heptane, pure heptane?
A B
C
D
What is [Chlorophyll a] in ?
ì Remember: The extract is a mix of compounds… the standard only contains chlorophyll!
Use the data from week 1 to find [chlorophyll a]:
ì from Absorbance…
ì from Fluorescence…
ì Which method is be2er to calculate concentra=on?
Beer’s Law Eqn: A = εCl
Calibra0on Curve
at 2 λs… low & high